Palaeoproteomic investigation of an ancient human skeleton with abnormal deposition of dental calculus

Detailed investigation of extremely severe pathological conditions in ancient human skeletons is important as it could shed light on the breadth of potential interactions between humans and disease etiologies in the past. Here, we applied palaeoproteomics to investigate an ancient human skeletal individual with severe oral pathology, focusing our research on bacterial pathogenic factors and host defense response. This female skeleton, from the Okhotsk period (i.e., fifth to thirteenth century) of Northern Japan, poses relevant amounts of abnormal dental calculus deposition and exhibits oral dysfunction due to severe periodontal disease. A shotgun mass-spectrometry analysis identified 81 human proteins and 15 bacterial proteins from the calculus of the subject. We identified two pathogenic or bioinvasive proteins originating from two of the three “red complex” bacteria, the core species associated with severe periodontal disease in modern humans, as well as two additional bioinvasive proteins of periodontal-associated bacteria. Moreover, we discovered defense response system-associated human proteins, although their proportion was mostly similar to those reported in ancient and modern human individuals with lower calculus deposition. These results suggest that the bacterial etiology was similar and the host defense response was not necessarily more intense in ancient individuals with significant amounts of abnormal dental calculus deposition.


Palaeoproteomic investigation of an ancient human skeleton with abnormal deposition of dental calculus
Yoko Uchida-Fukuhara 1,2* , Shigeru Shimamura 3 , Rikai Sawafuji 2,4 , Takumi Nishiuchi 5 , Minoru Yoneda 6 , Hajime Ishida 4,7 , Hirofumi Matsumura 8 & Takumi Tsutaya 2,9* Detailed investigation of extremely severe pathological conditions in ancient human skeletons is important as it could shed light on the breadth of potential interactions between humans and disease etiologies in the past.Here, we applied palaeoproteomics to investigate an ancient human skeletal individual with severe oral pathology, focusing our research on bacterial pathogenic factors and host defense response.This female skeleton, from the Okhotsk period (i.e., fifth to thirteenth century) of Northern Japan, poses relevant amounts of abnormal dental calculus deposition and exhibits oral dysfunction due to severe periodontal disease.A shotgun mass-spectrometry analysis identified 81 human proteins and 15 bacterial proteins from the calculus of the subject.We identified two pathogenic or bioinvasive proteins originating from two of the three "red complex" bacteria, the core species associated with severe periodontal disease in modern humans, as well as two additional bioinvasive proteins of periodontal-associated bacteria.Moreover, we discovered defense response system-associated human proteins, although their proportion was mostly similar to those reported in ancient and modern human individuals with lower calculus deposition.These results suggest that the bacterial etiology was similar and the host defense response was not necessarily more intense in ancient individuals with significant amounts of abnormal dental calculus deposition.
Ancient human skeletons sometimes show abnormal and extremely severe pathological conditions that could be rarely observed in modern human populations 1,2 .These extreme cases emphasize both human resilience and vulnerability to disease in the absence of modern healthcare interventions 3,4 .Humans and pathogens coevolved and various ancient pathogens are not equivalent to their contemporary descendants 5,6 .Ancient severe pathological conditions that cannot be seen today could have existed due to the lack of modern medical interventions or different bacterial etiologies.Detailed investigation of these extreme cases would be important as they shed light on the breadth of potential interactions between humans and diseases, and reveal differences between past disease etiologies and present-day pathogens.
In this study, we used palaeoproteomics to investigate the etiology of and host resilience to periodontal disease in an ancient human skeleton showing abnormal deposition of dental calculus with severe periodontal disease.Dental calculus is a calcified oral plaque that promotes periodontal disease 7 and is habitually removed in modern dental care.In contrast, abnormal depositions of dental calculus, where a large calculus deposition entirely covers the occlusal surface of at least one tooth, could be occasionally observed in ancient human skeletons.

The subject individual, HM2-HA-3
HM2-HA-3 is a female skeleton, aged 34-54 years at death, excavated in 1992 from the Hamanaka 2 site (Fig. 1) on Rebun Island, Hokkaido, Japan 23 .The most notable feature of this individual is the abnormal deposition of large amounts of dental calculus (Fig. 1 10 ).The morphological characteristics of this individual have been previously described in detail 10 .Briefly, most skeletal elements of HM2-HA-3 were missing; only a part of the cranium, an upper limb, and trunk bones were present, though the mandible and maxilla, including erupted teeth were well-preserved.Heavy deposits of dental calculus were present, especially on the right side of the dentition.These calculus deposits are predominantly located above the cementoenamel junction, a feature of supragingival calculus.These deposits were primarily found on the right upper second and third molars (Fig. 1).The occlusal surfaces of these molars are completely covered by calculus deposits and present a non-smooth surface.
HM2-HA-3 also exhibits extreme oral pathological conditions.Caries are not present in any of the remaining teeth but HM2-HA-3 presents apical lesions with cementum hyperplasia, rounded cavities in the root apex, and severe periodontal disease including resorption of the alveolar process 10 .Periodontitis-related horizontal alveolar bone resorption was prominent in HM2-HA-3, and the mandibular right molars had been completely lost with severe resorption of the crest.This individual would likely have suffered from the periodontal disease since the relatively early stages of her life, when the right side of her jaws would have become almost completely unusable for masticatory function 10 .As a result, HM2-HA-3 showed severe tooth wear on her left teeth, which were not covered by calculus.Furthermore, alveolar bone resorption at the root branch was observed on the upper right side, suggesting the presence of endodontic-periodontal disease.Abnormal calculus deposition would have facilitated periodontal tissue collapse in the same region.Taken together, these conditions show that normal masticatory function would have been impaired in this individual.
HM2-HA-3 was found in an archaeological site belonging to the Okhotsk culture.The Okhotsk culture was distributed along southern Sakhalin Island, the northeastern coast of Hokkaido, and the Kuril Islands during the fifth to thirteenth centuries 24 .The Okhotsk people predominantly subsisted on fishing, and it is estimated that marine foods comprised more than 80% of their dietary protein intake 25,26 .Although a few crop remains have been excavated from Okhotsk sites 27 , it is believed that plant horticulture was not practiced in the Okhotsk culture 24 .Because of their low carbohydrate intake, the caries rate of Okhotsk people was remarkably lower than

Dental calculus proteome
We identified a total of 96 protein groups from the dental calculus of HM2-HA-3, excluding keratins and common laboratory contaminants.Of these, 81 and 15 protein groups originated from humans (Table 2) and bacteria (Table 3), respectively.The calculus displayed a high (i.e., 92.1%) OSSD score, suggesting good protein preservation 20 .The peptide deamidation rates, the approximate proxy for ancient protein authenticity 36,37 , derived from the four fractions ranging between 38.7-54.8% and 30.7-37.7% for asparagine and glutamine in human proteins, respectively (Supplementary Table S1).As the deamidation rate of modern proteins is typically below 20%, the human proteins identified in the dental calculus of HM2-HA-3 would originate from ancient times 13 .In contrast, bacterial proteins showed lower asparagine and glutamine deamidation rates (4.9-23.2% and 4.2-24.0%,respectively) (Supplementary Table S1).The number of asparagine and glutamine residues in the identified bacterial proteins was below 8, the precise deamidation rates could thus not be calculated.
The identified human proteins were classified with GO term using the PANTHER software 38 .Among the assigned protein class, 13.9% represented the "defense/immunity." Among the proteins categorized in this class, peptidoglycan recognition protein 1 was one of the innate immune system proteins and functions to directly kill bacteria by recognizing and cleaving peptidoglycans on the bacterial wall 39 .Neutrophil elastase is among the antimicrobial peptides abundant in the saliva and gingival crevicular fluid in the oral cavity and is involved in local defense mechanisms 40 .
We identified a total of 15 proteins from 13 bacterial taxa from the calculus.Eight of these originated from six bacterial taxa that are reportedly associated with periodontal disease in modern patients (Table 3).We identified two of the three "red complex" bacteria, the most notable core bacterial species in the severe form of periodontal disease (Porphyromonas gingivalis and Treponema denticola).In addition, among the identified bacterial taxa, Selenomonas sputigena and Fretibacterium fastidiosum are reportedly associated with severe periodontal disease in modern humans 41,42 , while Actinomyces dentalis and Actinomyces israelii were identified in patients with severe periodontal disease 43 .Pathogenic factors of P. gingivalis, a proteolytic enzyme of Lys-gingipain W83 and Mfa1 were identified in the calculus with well-annotated MS2 spectra (Supplementary Figure S2) 44 .Moreover, pathologically invasive proteins, such as T. denticola flagellar filament 33-kDa core protein, F. fastidiosum flagellin, and S. sputigena flagellar filament 33-kDa core protein, were also identified with well-annotated MS2 spectra (Supplementary Figure S2).These flagellar proteins are associated with bacterial motility and could initiate immune responses by interacting with toll-like receptor 5 in the host [45][46][47][48] .We could not identify any bacterial taxa and dental caries-associated proteins.Our BLAST search indicated that the peptide sequences of these periodontal disease-associated bacterial proteins only occur in certain bacterial genera (Supplementary Table S2).
We compared the protein groups or bacterial taxa identified in the dental calculus of HM2-HA-3 with those identified in a previous palaeoproteomic analysis of ancient human dental calculus from medieval Dalheim, Germany as well as those of modern European patients with periodontitis and dental caries 13 .As presented in Fig. 3, 49.4% (40/81) of the human proteins and 69.2% (9/15) of the bacterial taxa identified in HM2-HA-3 calculus were also identified either in Dalheim or modern calculus 13 .with the common bacterial taxa being P. gingivalis, A. israelii, Actinomyces sp.HMT 414, and Corynebacterium matruchotii.Bacterial species unique to HM2-HA-3 included S. sputigena, Actinomyces sp.HMT 169, Selenomonas sp.HMT 892, and Campylobacter gracilis 49 .
Finally, we performed a proteomic analysis of a rib bone sample of HM2-HA-3 to investigate the potential presence of systematic diseases.We identified a total of 59 human proteins, most of them being bone proteins (Supplementary Table S3).We could not identify any systematic disease-associated protein.

Discussion
The palaeoproteomic analysis of abnormally deposited dental calculus conducted here provided molecular insights into the pathological conditions of the oral cavity of HM2-HA-3.We identified both pathogenic factors and bioinvasive proteins (i.e., Lys-gingipain W83, Mfa1, flagellin, and flagellar filament 33-kDa) from bacterial taxa reportedly associated with periodontal disease in modern patients.The identification of these proteins provides molecular support for the periodontal disease of this individual originally diagnosed based solely on physical characteristics.These bacterial proteins are associated with periodontal disease pathogenesis and development as well as with the secretion of inflammatory cytokines [45][46][47][48]50 .
Of the 13 bacterial taxa identified from the calculus of HM2-HA-3, seven (53.8%) are reportedly associated with periodontal disease in modern clinical medicine (Table 3), in particular, two of the three red complex bacterial taxa.Proteins from the red complex bacteria have frequently been identified in both modern and ancient human dental calculus samples 13,15,51,52 .In this study, the pathogenic protein of P. gingivalis and bioinvasive protein of T. denticola were confidently identified 53 , providing direct evidence of red complex bacterial involvement in periodontal disease etiology.Although the involvement of the remaining seven bacterial taxa in the etiology of periodontal disease remains unclear, our results confidently indicate that periodontal disease bacterial etiology in HM2-HA-3 was similar to that in modern patients.
The presence of various host defense response proteins suggests that HM2-HA-3 was subjected to pathological stress and the resulting inflammation, at least during dental calculus deposition.However, the identified host defense proteins were nonspecific (e.g., lactotransferrin, immunoglobulin kappa constant, and prolactininducible protein) and mostly similar to those identified in other ancient individuals with significantly lower calculus deposition (Supplementary Fig. S3) 13 .Moreover, our PANTHER analysis revealed that the "immune system process" comprised 6.9% of the total processes assigned to the identified host proteins in the HM2-HA-3  4).This proportion is rather lower compared to those in the calculus samples from medieval Dalheim (8.1%) and modern patients suffering from moderate to moderate/severe periodontal disease (10.7%) 13 .Furthermore, the proportion of the "defense/immunity protein" class was also lower in the calculus of HM2-HA-3 (13.9%) than that in the modern dental calculus (20.8%) and was somewhat higher than that in the calculus sample of medieval Dalheim (10.4%) 13 .These results imply that host defense response to oral pathological stress was not necessarily higher in HM2-HA-3, who exhibited significant amounts of calculus deposits and severe masticatory dysfunction, relative to modern periodontitis patients and medieval individuals with lower calculus deposition.
Although palaeoproteomics provides molecular evidence on the bacterial etiology of and host defense response to periodontal disease, the cause of the abnormal calculus deposition in HM2-HA-3 remains unclear.Diet is often cited as a cause for calculus deposition 54 , but this cause is unlikely for HM2-HA-3.Stable isotope analysis showed that HM2-HA-3 had a similar diet to other individuals from the Hamanaka 2 site and other individuals from Hamanaka 2 site displayed little or no calculus deposition (Fig. 2).Abnormally high amounts of calculus deposition could occasionally be seen in modern patients, but the underlying cause is unidentifiable in most cases 55,56 .At least, this individual would not have a routine tooth cleaning habit during the period of calculus deposition.Furthermore, as the HM2-HA-3 bone proteome did not contain disease-indicative proteins, calculus deposition unlikely occurred as a systemic disease byproduct.
HM2-HA-3 is the first individual among the ancient human skeletons from Asia with a bacterial proteome studied in detail.Therefore, in this study, we used for comparison of previously published proteome results on calculi from individuals in Europe 12 .Almost all published bacterial proteomes of modern and ancient dental calculus originate from Europe 12,14 .As the regional differences in the human oral bacterial composition have been suggested 57 , accumulating data on dental calculus bacterial proteome outside Europe would be required.

Materials and methods
Detailed procedures regarding sample collection and analyses are described in the Supplementary Materials and Methods.A brief summary is shown below.

Sampling
Dental calculus was collected from the lower right first incisor of HM2-HA-3 (Supplementary Figure S1), with the method described previously 58 .Given the small variability in bacterial composition in calculus obtained  59 , we assume that this sample had a representative bacterial composition as would be obtained from the abnormally deposited calculus present on the molars (Fig. 1).Rib bones were also sampled for palaeoproteomic and isotope analyses.

Proteomics
Protein extraction from 15 mg of dental calculus was performed using modified ultrafiltration and single-pot solid-phase-enhanced sample preparation (SP3) methods for ancient protein analysis 60,61 .Protein extraction  from 20 mg rib bone was performed using modified ultrafiltration method 62 .Following the guidelines for palaeoproteomics 18 , the entire extraction process was carried out in a clean laboratory dedicated to ancient biomolecules built at the Graduate University for Advanced Studies, Japan.We obtained four fractions of the calculus sample (i.e., supernatant and pellet fractions from each of the ultrafiltration and SP3 methods) and two fractions (i.e., supernatant and pellet) of the bone sample along with experimental blanks.Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of dental calculus was performed using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific) at Japan Agency for Marine-Earth Science and Technology (JAMSTEC) with the conditions described in Nunoura et al. 63 .LC-MS/MS analysis of rib bone was performed using an Orbitrap QE Plus mass spectrometer (Thermo Fisher Scientific) at Kanazawa University with the conditions described in Ogura et al. 64 .RAW data files generated by LC-MS/MS were analyzed using the MaxQuant software version 2.0.1.0 65.Data of calculus were searched against the Oral Signature Screening Database (OSSD 20 ) for the first quality-assurance step and the electric Human Oral Microbiome Database (eHOMD 66 ) or entire human proteome (as of 2023-03-02) for the second protein identification step.Data of bone were searched against the entire human proteome.Because no food proteins were identified from dental calculus in a MaxQuant search against an entire Swiss-Prot database (as of 2021-08-20), we did not investigate food proteins.Comparative datasets were analyzed anew in the same manner 12 .Gene Ontology (GO) analysis of the human-derived proteins identified from the dental calculus of HM2-HA-3 was performed using PANTHER, version 14 38 .Python script reported by Mackie et al. 14 was used to calculate asparagine and glutamine deamidation rates.All subsequent data analyses were performed using R, version 4.2.2 (R Core Team, 2022).

Radiocarbon dating and stable isotope analysis
Collagen was extracted from a rib bone of HM2-HA-3 to conduct radiocarbon measurement and carbon and nitrogen stable isotope analysis, based on the method described previously 67 .Carbon and nitrogen stable isotopes were measured using elemental analyzer-isotope ratio mass spectrometry (EA-IRMS) at the University Museum, the University of Tokyo (UMUT).
Radiocarbon concentrations were measured using accelerator mass spectrometry (AMS) at UMUT.Radiocarbon age was calibrated against atmospheric and marine calibration curves (IntCal20 and Marine20 68,69 ) and with the local marine reservoir effect 70 using OxCal, version 4.4 71 .

Figure 1 .
Figure 1.(a) Map of Rebun Island.The map was drawn with the packages ggplot2 (version 3.4.0)and rnaturalearth (version 0.3.2) on version 4.2.2 of R. (b) Right buccal aspect of the HM2-HA-3 maxilla and mandible.A red arrow indicates the sampled calculus (i.e., from the lower right permanent first incisor).

Figure 3 .
Figure 3. Venn diagrams of (a) human proteins and (b) bacterial taxa identified in the ancient dental calculus of HM2-HA-3 (this study) as well as in the dental calculus samples from medieval Dalheim and modern patients 12 .

Figure 4 .
Figure 4. Results of PANTHER (a) biological process and (b) protein class analysis of protein groups identified in the dental calculus of HM2-HA-3 (this study) as well as in the dental calculus samples from medieval Dalheim and modern patients 12 .

Table 1 .
Results of stable isotope analysis and radiocarbon measurement.Previously reported data from other skeletal individuals from the Hamanaka 2 site are also shown.

ID sex Age (y) Element %C %N δ 13 C δ 15 N C/N 14 C age (BP) Reference
Figure 2. Carbon and nitrogen stable isotopic results of faunal and human bone collagen.Vol:.(1234567890)

Table 2 .
Human protein groups identified in the dental calculus of HM2-HA-3.
from different oral positions within an individual

Table 3 .
Oral bacterial protein groups identified in the dental calculus of HM2-HA-3.